Manipulations of the Immune Response

Parameth Thiennimitr, M.D., Ph.D.

Department of Microbiology
Faculty of Medicine, Chiang Mai University
2016

Learning Objectives
1. Immunotherapy VS. Vaccination
2. Important immunomodulators
3. Gut microbiota and Immune modulation
4. Active and passive immunizations
5. Immunological memory and vaccination
6. Immunological basis of each types of vaccines

Manipulation of the Immune Response

Treatment of
unwanted immune
response

Stimulate protective
immune response
Passive

Immunotherapy

Active

Vaccination

Immunotherapy

Immunotherapy
Treatment of unwanted immune

response
Graft rejection

Hypersensitivity
Autoimmunity

Immunomodulators
Traditional
Pharmaceutical
Medicine

Biopharmaceutical
Medicine
= Biologics

(Chemically synthesized)
(Biologically synthesized)

Chemically-Synthesized
Immunomodulators

Immunosuppressive Agents
(3) T cell proliferation
inhibition agents

(4) T cell signaling

inhibition agents

T cell
cytokines

Inflammatory
cells

mediators

Tissue damage from

(2) Anti-mediators:
antihistamine

inflammation

(1) Anti-inflammation agents

(1) Anti-Inflammation Agent:

Corticosteroid
steroid

Cell membrane

Steroid
receptor
(SR)

Annexin-1 Cytoplasm
(anti-inflammation)
SR
translocation

Hsp90 Immunophilin

dimerization

Regulatory
element

Nucleus

Control Gene
Expression
RE

Proinflammatory
cytokines

Corticosteroid
Proinflammatory
cytokines & enzymes

Annexin-1
(anti-inflammation)

- COX2 (cyclooxygenase-2)

- Inhibits COX2

- iNOS (inducible nitric oxide

- Inhibits phospholipase A2,

synthase)
- Most proinflammatory
cytokines:
IL-1, IL-6, IL-13, GM-CSF,

IFN-, TNF-

PLA2 (PGs, Leukotrienes

production)
- Promotes neutrophil
detachment and apoptosis
- Decreases neutrophil

infiltration

Wide range of
steroid side effects!

Neurohormonal disturbance

Glaucoma and cataract

Myopathy
Hypertension
Osteoporosis
Skin thinning
Hyperglycemia

Infection

Adrenal insufficiency
(corticosteroid
withdrawal)

(2) Anti-Mediators

Antihistamine
H1-receptor on
endothelium

H1-receptor
antagonist
Allergic rhinitis

histamine

1st generation
Cross blood brain
barrier

Cross blood
brain barrier

(High sedative effect)

(less sedative)

Examples:

Degranulation
of mast cell

2nd generation

Examples:

- Chlorpheniramine

- Loratadine

- Diphenhydramine

- Fexofenadine

(3) T cell Proliferation Inhibition Agents:

DNA Alkylating Agents
6-thioguanine
(purine analog)

Compete with
inosine
monophosphate

Blocking de novo
synthesis of AMP
and GMP

Azathioprine
Mycophenolic
acid

Inhibit inosine
monophosphate
dehydrogenase

Mycophenolate
mofetil
Phosphoramide
mustard

Cyclophosphamide
(High toxicity e.g.
hemorrhagic cystitis)

Inhibit
DNA
synthesis

DNA alkylating
agent

Clinical uses:
High dose: cancer
Low dose : autoimmunity

Kill all
dividing
cells!

(4) T cell Signaling Inhibition Agents:

Immunophilin binding agents
(2) mTOR Inhibitor:
Rapamycin

APC

Immunophilin

IL-2R

TCR

IL-2

ITAMS

(1)
Calcineurin
Inhibitor:

mTOR

Calcineurin

Cyclosporin A

IL-2
mRNA

P
Immunophilin

P
NFAT
(inactive)

NFAT = nuclear factor of

activated T cells
mTOR = mammalian
target of rapamycin

Cytokine
genes
NFAT
(active)
nucleus

T cell

Biologically-Synthesized
Immunomodulators

Biologics
A newer type of therapeutic compounds
- Genetically engineered natural proteins from human genes
- Inhibit specific component of the immune system
Monoclonal
antibodies

Protein
fragments

BIOLOGICS

Cytokines

Polyclonal
antibodies

Whole cells

Inhibition of Lymphocyte Function by Antibody:

Polyclonal VS. Monoclonal Antibodies

More Specific, Less Direct Toxicity Compare to Drug
1. Polyclonal antibodies (anti-lymphocyte globulin)

Rabbit anti-human
polyclonal antibodies

Human
lymphocytes

Less specific,
More allergic
(rabbit protein)

2. Monoclonal antibodies
+
AntibodyMyeloma cell
producing B cells
(plasma cells)

Hybridoma
cells

Selection for the

desired clone (Ab)
One = Mono

More specific,
Less allergic

Engineered monoclonal antibodies to reduce immunogenicity

-Omab

-Ximab

-Zumab

Highly
immunogenic in
human

Mouse Variable
region +
Human Constant
region

Mouse Hypervariable
region +
Human constant
region

-Umab

Examples of Clinical Usage of Monoclonal Antibodies

Generic Name

Specificity

Mode of action

Clinical Use

Rituximab

Anti-CD20

Kill B cells

Lymphoma

Alemtuzumab

Anti-CD52

Kill lymphocytes

leukemia

Muromonab
(OKT3)

Anti-CD3

Inhibits T-cell
activation

Kidney
transplantation

Adalimumab

Anti-TNF-

Inhibit inflammation
induced by TNF-

Rheumatoid
arthritis

Omalizumab

Anti-IgE

Remove IgE

Asthma

Efalizumab

Anti-4 integrin

Block lymphocyte
trafficking

Multiple sclerosis

Ipilimumab

Anti-CTLA-4

Increase CD4+ T-cell

response

Metastatic
melanoma
(Cancer
immunotherapy)

Monoclonal Antibodies in Cancer Immunotherapy

NK cells use
CD16 to bind
antibody Fc

NK cells kill
tumor cell

(1) Tumorspecific antibody

tumor cell

Antibodies bind to the

tumor cell

(3) Tumor-

(2) Tumorspecific antibody

conjugated with
toxin

tumor cell

Toxin kill
tumor cell

specific
antibody
conjugated with
radionucleotide

tumor cell

Radiation kill
tumor cells

Tumor Vaccine by induction of CD8+ T cells

Tumor
antigen

Plasmid
expressing
tumor antigen

Vaccination

Tumor cell

Directly put tumor

antigen in to APC

APC with the

tumor antigen

Tumor cell

Destroy
tumor cell
Proliferation of tumorspecific CD8+ T cell

Tumor-specific
CD8+ T cell

Tumor Vaccine by Enhancing CD8+ T cell Function

Tumor cell

Activate
T-cell function

T-cell
proliferation

Destroy
tumor cell

Tumor-specific CD8+ T cell

Plasmids

B7
Vaccination

Tumor cell that can

produce B7 and IL-2

CD8+ T
cells

IL-2

Adoptive T-cell Transfer in Cancer Immunotherapy

IL-2

Modification of tumorspecific CD8+ T cell in vitro

Isolate
lymphocytes
from blood or
tumor infiltrate

(Adoptive Immunity)

Patient with
tumor

Tumor
regression

Proliferation of tumorspecific CD8+ T cells

The Newest Way to Manipulate

the Immune System

Were our microbes

human

Ley R., et al. Cell, 2006 (Picture from www.nature.com)

microbiota

Human colon contains the great numbers & variety of microbiota

Beneficial Roles of Gut Microbiota

Gut lumen

pathogens

1. Colonization resistance

Gut
microbiota

Dietary fiber

Short chain
fatty acid

Gut epithelium

3. Maintaining
intestinal
barrier
integrity

2. Regulation of gut
immune homeostasis

Gut lamina propria

Immune cells

4. Providing host
some nutrients

Gut microbiota
educate our
immune system
for proper
development,
maturation and
homeostasis

Manipulation of Gut Immune System via

Gut Microbiota Changing

SYNBIOTICS

PREBIOTICS

PROBIOTICS

Non-digestible compound that

stimulate growth of probiotics

Live bacteria that benefit host

e.g. Xylooligosaccharides

e.g. Lactobacillus, Bifidobacterium

Example of Probiotic Bacteria Use in Food Allergy

Administration of
probiotic
Lactobacillus
rhamnosus
in early life

Cows milk allergy

in children
(Aberrant gut immune
response to protein in
cows milk)

Reviewed by Cosenza L, et al, Benef Microbes, 2015

TH1/TH2
balance
Increases oral tolerance
to cows milk protein

Decreases risk and severity of

Cows milk allergy

Vaccination

Vaccination
The Origin of Immunology

Historian Thucydides
430 BC
The plague of Athens

Variolation

Vaccination

in China and India

1400-1500s

Edward Jenner
1700-1800s

..for the same man was

never attacked twicenever at least fatally

..inhalation of dried
small pox pustule
promotes longlasting protection

The Father of Immunology

The worlds first vaccine

(smallpox vaccine)

Edward Jenner (1749-1823)

An English Physician-Scientist

The Father of Immunology (Vaccination)

Vaccination
Form Cow (vacca) to Human
May 14, 1796
Protection
Jenner inoculated
an 8-year-old boy,
James Phipps

Deadly human
smallpox

Cowpox
(non-deadly)

A young dairy maid

(Sarah Nelms)

Cowpox lesion on
her hand

Vaccination against human smallpox with cowpox

share
common
antigens

smallpox
virus

Antibody to
cowpox
virus
antigens
neutralize
smallpox

vaccination
cowpox
virus

Antibody to
cowpox
virus
antigens

Vaccine Strategies:
Application of Immunological Memory

Innate
immunity

1Adaptive
immunity

Protective
immunity

Immunological
memory (Im)

2Adaptive
immunity

More
antibody

Faster

More T
cell
Second
infection

Repeat infection

Vaccination

Im

Secondary response

Primary response

First
infection

Protective
immunity

Immunological
memory (Im)

Primary Immune Response Yields

Both Effector & Memory T cells
Effector T cell

Nave T cells
activated by
pathogen

Memory T cell

Effector >
memory T cells

Primary Immune Response Yields

Both Effector & Memory B cells

Nave B cells
activated by
Memory B cell
pathogen and TFH cell
(Follicular helper T cell)

>

memory
pathogen-specific
B cells

Primary VS. Secondary Immune Response

pathogen

Faster and
Stronger

antibody
innate

T cell

memory

Primary

Secondary

Less effector cells

More effector cells

Delay

Immediately

Mixed affinity of Ab

High affinity of Ab

High activation threshold Low activation threshold

Innate works alone then
adaptive

Innate and adaptive

work together

Vaccination increases Antibody Quality and Quantity

Antibody affinity

Antibody amount

Vaccine Strategies: Active VS. Passive

Active immunization

Passive immunization

Vaccine

Antibody
(anti-sera)
- Need time to develop
- Long-life vaccine
(pathogen)-specific
antibody and T cells

- Ready to use with life

threatening diseases
- Short-life

Passive Immunization (Anti-Sera)

Specific
antibody to
severe diseases:
rabies, tetanus,
hepatitis etc.

Snake
antivenom

Pool of human
Immunoglobulins:
in primary immunodeficiency diseases

rabies

Features of Effective Vaccines

Neutralizing
antibody

Safe

Long lasting
protection

Low cost
Few
side-effects

Protective
T cells
Ease to
administration

7 Major Types of Vaccine

1. Live-attenuated vaccines

2. Inactivated (killed) vaccines

3. Subunit vaccines
4. Toxoid

5. Conjugate vaccines
6. DNA vaccines
7. Recombinant vector vaccines

(1) Live-Attenuated Vaccines

Lifelong immunity
Specific
CD8+ T cell
Virulent strain
Specific
antibody
destroy

Avirulent strain

Live- Attenuated

Not safe in an
immunocompromised
host!!

Virulent strain

How to make a Live-Attenuated Vaccine

I) Finding avirulent strains

Smallpox virus

Cowpox virus
= avirulent in
human

How to make a Live-Attenuated Vaccine

II) Grow virus in different host cells

Cannot infect human cell

Propagate
human virus in
the human
cells

Human cell

Monkey cell

Transfer
human virus
into monkey
cells

Human virus mutated

(adapted) to live in
monkey cell

Human cell
Avirulent human virus

Monkey cell

How to make a Live-Attenuated Vaccine

III) Genetic engineering (Recombinant DNA tech.)
Immunogenic but avirulent

Pathogenic
strain

Virulence
gene deletion

Virulence
gene

Virulence gene
mutation

Examples of Live-Attenuated Vaccines

Live-attenuated vaccine

Against

Bacillus Calmette-Guerin (BCG)

Tuberculosis

Oral polio vaccine (OPV, Sabin)

Poliomyelitis

Mumps-Measles-Rubella (MMR)

Mumps,
measles, rubella

Oral typhoid vaccine

Typhoid fever

Nasal influenza vaccine

Influenza

Chickenpox vaccine

Chickenpox

Rotavirus vaccine

Rotavirus

(2) Inactivated (killed) Vaccines

Short-life immunity
Low immunogenic
(require boosting)
Cannot
multiply

Specific
CD8+ T cell

Virulent microbe
Specific
antibody

inactivation

destroy

Dead microbe

Inactivated

Safe in
immunocompromised
host
Virulent microbe

Examples of Inactivated (killed) Vaccines

Inactivated (killed) vaccine

Against

Rabies vaccine

Rabies

Inactivated polio vaccine

(IPV, Salk)
Japanese encephalitis vaccine
(JEV)
Inactivated influenza vaccine

Poliomyelitis

Japanese
encephalitis
Influenza

(3) Subunit Vaccines

Short-life immunity
Low immunogenic
(require boosting)
Cannot
multiply

Specific
CD8+ T cell

Virulent microbe
isolation

Specific
antibody

destroy

Only antigen
- protein
- polysaccharide

Safe in
immunocompromised
host

Example: Hepatitis A, B vaccines Virulent microbe

(4) Toxoid
Toxigenic domain

Core protein

Cannot
multiply

Microbial toxin

Neutralize
toxin

Core protein

Detoxified toxin

Low immunogenic
(require boosting)

Anti-toxin
antibody

Detoxification

Toxoid

Short-life immunity

Safe

Example: Tetanus toxoid (TT), Diphtheria toxoid (DT)

(5) Conjugate Vaccines

(Polysaccharides + Protein Carrier)
Young children
Age < 2 years
polysaccharides
= T independent
antigen (TI)

T cell
conjugate
vaccine

Crosslinking

polysaccharides

Poor
B cell

Protein carrier
Specific antibody
production without
help of T cell
Example: Haemophilus influenzae
type b (Hib) vaccine

Less antibody

How does a conjugate vaccine work?

B-cell binds to polysaccharide
conjugated with protein
protein

Antigen processing by B-cell

polysaccharide

B-cell

Activated B-cell produces Ab to

polysaccharide

B-cell presents peptide

antigen to T-cell
THcell

Signals from T-cell activate B-cell

(6) DNA Vaccines

Insertion of microbial DNA directly into host nucleus

Microbial
antigen-specific
immunity
Microbial DNA
Host cell

Host cells express

microbial antigens

(Preclinical trial)
e.g. Influenza, herpes

(7) Recombinant Vector Vaccines

Host cell

Microbial antigenspecific immunity

+
Microbial DNA

Vectors:
Bacteria, Virus

Host cells express

microbial antigens

(Preclinical trial)
e.g. HIV, rabies,

Adjuvant
= Substance that enhance immunogenicity of vaccine

Adjuvants

Freunds adjuvant

Mechanisms of Action
Delayed antigen release
(Enhance phagocytosis)

Alum (aluminum hydroxide)

Delayed antigen release

(Enhance phagocytosis)

Immune stimulatory
complexs (ISCOMs)

Deliver antigen to
cytoplasm

Systemic Vaccine Routes

subcutaneous
(ID): high immune
cells & lymphatic
vessels
45

Intramuscular
(IM): adjuvantcontaining
vaccine
Epidermis

10

Intradermal
(ID): slow
absorption

Dermis
Subcutane
ous tissue

Muscle

Local Vaccine Routes

1. Mimic natural infection
2. Stimulate local
(mucosal) immunity

Nasal
Oral

References
1.

Abbas Ak, Litchman AH and Pillai S. Cellular and Molecular Immunology, 8th
ed. Philadelphia:Elsevier Sauder; 2014

2.

Murphy KP. Janeways Immunobiology. 8th ed. New York: Garland Science;
2012

3.

Parham P. The Immune System. 4th ed. New York: Garland Science; 2015

4.

http://www.niaid.nih.gov/topics/vaccines/understanding/pages/typesvaccines.a
spx

5.

Phillip D. Smith et al. Principles of Mucosal Immunology. 1st ed. London and
New York: Garland Science; 2013

6.

Cosenza L. et al. Bugs for atopy: the Lactobacillus rhamnosus GG strategy for
food allergy prevention and treatment in children, Benef Microbes.
2015;6(2):225-32. doi: 10.3920/BM2014.0158.